Composition for stimulating bone-formation and bone consolidation

ABSTRACT

The present invention relates to a composition for stimulating bone-formation and bone-consolidation, more particularly, to a composition for stimulating bone-formation and bone-consolidation by adding a material for stimulating bone-forming and bone-consolidation to the mixture of tripolyphosphate and water-soluble chitosan. The composition of the present invention can stimulate bone-formation and bone-consolidation in early stages.

FIELD OF THE INVENTION

The present invention relates to a composition for stimulatingbone-formation and bone-consolidation, more particularly; to acomposition for stimulating bone-formation and bone-consolidation byadding a material for stimulating bone-forming and bone-consolidation tothe mixture of tripolyphosphate and water-soluble chitosan.

BACKGROUND ART OF THE INVENTION

Bone-loss is often caused by a disease or a car accident recently, sothat supplementing bone-loss is importantly required.Bone-transplantation is one way to supplement bone-loss and morepreferably bone-filling composition is used. Bone-extension technique isperformed today to extend one's height or to correct undersized jaws,for which bone-filling composition is also required a lot.

Bone-extension technique is to stimulate bone-growth, especially growthin height, by stretching based on the theory that “Tension forcesstimulate histogenesis”. Bone-extension technique was first devised forthe growth of limb bones but has been widely used for jawbone extension.Jawbone extension method is one of techniques performed in the field ofcranial jaw facial surgery, which can improve facial ratio not bycutting bone but by moving facial bones gradually by fixingbone-stretching apparatus to retreated parts of jawbone and centralfacial form.

Bone-extension technique has been successfully used for supplementingthe loss of long bone since Ilizarov found out biomechanical elementsfor bone-extension (Ilizarov G A, J. Dis. Orthop. Inst., 48(1): 1, 1988;Ilizarov G A, Clin. Ortho., 239: 263, 1989; Ilizarov G A, Clin. Ortho.,238: 249, 1989). It is important for performing the successfulbone-extension to keep blood circulation in the part of bone-extensionwell and to fix external fixator stably to both sides of joint part ofcortical bone, resulting in the stimulation of bone-consolidation bygradual extension of bone (White S H, J. Bone Join Surgery, 72-B: 350,1990; White S H, Orthop. Clin. North. Amer., 22: 569, 1991; FishgrundJ., Paley D., Sulter D., Clin. Orthop., 301: 31, 1994).

The period of bone-consolidation depends on extension part of bones suchas facial bone or long bone, blood circulation condition, the age of apatient, etc. Bone-consolidation of craniofacial bone takes 3-5 weeksfor children and 6-12 weeks for adults after bone-extension, while ittakes 3-6 months in long bone regardless of age. Performingbone-extension for craniofacial bone has a couple of problems; one iscarrying high possibility of complications and the other is postponingthe return to normal life due to the long bone-consolidation time.Precisely, the treatment after bone-extension takes 2-4 months composingof latent phase, bone-extension phase and bon-consolidation phase.

According to Charls and Sailer's report, extending 1 mm a day showsstronger biochemical and physiological characteristics than extending2-3 mm a day (Carls & Sailer, J. Craniomaxillofac Surg., 94: 152, 1994).Ilizarov has also reported that extending 1 mm a day showed best resultswhile extending 0.5 mm a day caused premature bone-consolidation andextending 2 mm a day caused undesirable changes in extended tissues(Ilizarov, J. Dis. Orthop. Inst., 48 (1): 1, 1988; Ilizarov, Clin. Ortho239: 263, 1989). In addition, it has also been known that consecutiveextending causes the least damage in tissues but the best development ofcapillary vessels and bone-formation. Therefore, shortening the periodof bone-extension and bone-consolidation can contribute to preventpossible complications and to make a patient return to normal lifeearly. In order to shorten the period of bone-extension andbone-consolidation, bone-filling composition is used to stimulatebone-formation and bone-consolidation.

Meanwhile, autobone-graft, treated homograft, heterograft and bone graftsubstitute have been known to stimulate bone-formation. Autobone-graftis used for the treatment of joint-agglutination or non-agglutinationalfracture, or for avoiding damage and void caused by infection, tumor andoperation by supplementing bone cavity or bone loss. Transplantedautobone is well adsorbed, resulting in re-circulation of blood. At thistime, osteoprogenitor cells are differentiated to bony osteogenesiscells and the activation thereof stimulates bone-regeneration as well astreats bone-loss. However, autobone-graft has problems such aslimitation in the amount of extraction and high morbidity caused by thesecondary operation for the part of donation. Thus, bone morphogenicprotein or other bone-grafting substitute is used to inducebone-regeneration at extended sites. Bone morphogenic protein isregarded as the strongest bone-inducing material but is limited inclinical use because it is very expensive and hard to obtain.

Thus, the present inventors have been tried to find out a composition tostimulate bone-formation and bone-consolidation that is inexpensive andsuitable for human. As a result, we, the present inventors, haveprepared a composition by adding a material stimulating bone-formationand bone-consolidation to the mixture of tripolyphosphate and watersoluble chitosan, and have completed the present invention by confirmingthat the composition stimulates bone-consolidation in the early phaseand shortens the period of bon-generation by cutting bone-consolidationtime.

SUMMARY OF THE INVENTION

The present invention relates to a composition for stimulatingbone-formation and bone-consolidation, more particularly, to acomposition for stimulating bone-formation and bone-consolidation byadding a material for stimulating bone-formation and bone-consolidationto the mixture of tripolyphosphate and water-soluble chitosan. Thecomposition of the present invention can stimulate bone-formation andbone-consolidation in the early stage.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a photograph showing the performance of vertical osteotomy atleft jaw of a dog for bone-extension with chitosan, β ig-h3 and humanbone morphogenic protein (BMP-4),

FIG. 2 is a photograph showing a dual syringe containing water-solublechitosan, water-soluble chitosan containing β ig-h3, water solublechitosan containing BMP-4, and 5% tripolyphosphate,

-   -   A: 0.5 cc of water soluble chitosan, water soluble chitosan        containing β ig-h3, or water soluble chitosan containing BMP-4    -   B: 0.5 cc of 5% tripolyphosphate

FIG. 3 is a photograph showing a dog in the death-imminent state, sevenweeks after bone extension,

FIG. 4A is a photograph showing the extent of bone-consolidation in acontrol group that was measured with radio-assay five weeks after boneextension, particularly the control group was treated with onlytripolyphosphate and was induced to have 2.0 mm extension per day forfive days for the group,

FIG. 4B is a photograph showing the extent of bone-consolidation in acontrol group that was measured with radio-assay seven weeks after boneextension, particularly the control group was treated with onlytripolyphosphate and was induced to have 2.0 mm extension per day forfive days,

FIG. 5A is a photograph showing the extent of bone-consolidation in achitosan group that was measured with radio-assay 4 weeks after boneextension, particularly the group was treated with chitosan andtripolyphosphate, and was induced to have 2.0 mm extension per day for 5days,

FIG. 5B is a photograph showing the extent of bone-consolidation in achitosan group that was measured with radio-assay 7 weeks after boneextension, particularly the group was treated with chitosan andtripolyphosphate, and was induced to have 2.0 mm extension per day for 5days,

FIG. 6A is a photograph showing the extent of bone-consolidation in a βig-h3 group that was measured with radio-assay 4 weeks after boneextension, particularly the group was treated with water-solublechitosan containing β ig-h3 and tripolyphosphate, and was induced tohave 2.0 mm extension per day for 5 days,

FIG. 6B is a photograph showing the extent of bone-consolidation in a βig-h3 group that was measured with radio-assay 7 weeks after boneextension, particularly the group was treated with water-solublechitosan containing β ig-h3 and tripolyphosphate, and was induced tohave 2.0 mm extension per day for 5 days,

FIG. 7A is a photograph showing the extent of bone-consolidation in aBMP-4 group that was measured with radio-assay 4 weeks after boneextension, particularly the group was treated with water-solublechitosan containing BMP-4 and tripolyphosphate, and was induced to have2.0 mm extension per day for 5 days,

FIG. 7B is a photograph showing the extent of bone-consolidation in aBMP-4 group that was measured with radio-assay 7 weeks after boneextension, particularly the group was treated with water-solublechitosan containing BMP-4 and tripolyphosphate, and was induced to have2.0 mm extension per day for 5 days,

FIG. 8A is a graph showing the mineral density of bone 4 weeks afterbone extension for each group that was injected with chitosan only,water soluble chitosan containing β ig-h3 and water soluble chitosancontaining BMP-4 respectively, and was induced to have 2.0 mm extensionper day for 5 days,

FIG. 8B is a graph showing the mineral density of bone 7 weeks afterbone extension for each group that was injected with chitosan only,water soluble chitosan containing β ig-h3 and water soluble chitosancontaining BMP-4, and was induced to have 2.0 mm extension per day for 5days,

FIG. 9A is a photograph showing the histological section of a controlgroup 4 weeks after bone extension, particularly the group was injectedwith tripolyphosphate only and was induced to have 2.0 mm extension perday for 5 days,

-   -   Arrows: the first cutting parts

FIG. 9B is a photograph showing the histological section of a controlgroup 7 weeks after bone extension, particularly the group was injectedwith tripolyphosphate only and was induced to have 2.0 mm extension perday for 5 days,

-   -   Arrows: the first cutting parts

FIG. 9C is a photograph showing the new bone-formation at the edge ofbone extension region in a control group injected tripolyphosphate onlyand induced to have 2.0 mm extension per day for 5 days, which wasconfirmed by hematoxylin & eosin staining,

-   -   A: fibrous tissue, B: osteoblasts

FIG. 10A is a photograph showing the histological section of a BMP-4group 4 weeks after bone extension, particularly the group was injectedwith water soluble chitosan containing BMP-4 and tripolyphosphate, andwas induced to have 2.0 mm extension per day for 5 days,

-   -   Arrows: the first cutting parts

FIG. 10B is a photograph showing the histological section of a BMP-4group 7 weeks after bone extension, particularly the group was injectedwith water soluble chitosan containing BMP-4 and tripolyphosphate, andwas induced to have 2.0 mm extension per day for 5 days,

-   -   Arrows: the first cutting parts

FIG. 10C is a photograph showing that the center of the extended regionwas filled with osteoblasts and fibrous tissues, which was confirmed byhematoxylin & eosin staining with the histological section of A,

FIG. 10D is a photograph showing that the center of the extended regionwas filled with osteoblasts and fibrous tissues, which was confirmed byhematoxylin & eosin staining with the histological section of B,

FIG. 11A is a photograph showing the histological section of a β ig-h3group 4 weeks after bone extension, particularly the group was injectedwith water soluble chitosan containing β ig-h3 and tripolyphosphate, andwas induced to have 2.0 mm extension per day for 5 days,

-   -   Arrows: the first cutting parts

FIG. 11B is a photograph showing the histological section of a β ig-h3group 7 weeks after bone extension, particularly the group was injectedwith water soluble chitosan containing β ig-h3 and tripolyphosphate, andwas induced to have 2.0 mm extension per day for 5 days,

-   -   Arrows: the first cutting parts

FIG. 11C is a photograph showing the new bone-formation over the wholeextended region, which was confirmed by hematoxylin & eosin stainingwith the histological section of B,

FIG. 12A is a photograph showing the histological section of a chitosangroup 4 weeks after bone extension, particularly the group was injectedwith chitosan and tripolyphosphate, and was induced to have 2.0 mmextension per day for 5 days,

-   -   Arrows: the first cutting parts

FIG. 12B is a photograph showing the histological section of a chitosangroup 7 weeks after bone extension, particularly the group was injectedwith chitosan and tripolyphosphate, and was induced to have 2.0 mmextension per day for 5 days,

-   -   Arrows: the first cutting parts

FIG. 12C is a photograph showing the new bone-formation over the wholeextended region, which was confirmed by hematoxylin & eosin stainingwith the histological section of A,

DETAILED DESCRIPTION OF THE INVENTION

To achieve the above object, the present invention provides acomposition for stimulating bone-formation and bone-consolidationprepared by adding a material stimulating bone-formation andbone-consolidation to the mixture of tripolyphosphate and water-solublechitosan.

The present invention also provides a use thereof for stimulatingbone-formation and bone-consolidation in the early phase.

Further features of the present invention will appear hereinafter.

The present invention provides a composition for stimulatingbone-formation and bone-consolidation prepared by adding a materialstimulating bone-formation and bone-consolidation to the mixture oftripolyphosphate and water-soluble chitosan.

The composition for stimulating bone-formation and bone-consolidation ofthe present invention is prepared by adding a material stimulatingbone-formation and bone consolidation to the mixture of tripolyphosphateand water-soluble chitosan. β ig-h3, bony morphogenic protein, TGF-β,FGF, IGF-1 and PDGF are the examples for the material to stimulatebone-formation and bone consolidation, but the examples are not alwayslimited thereto. In the preferred embodiments of the present invention,β ig-h3 and BMP-4 were used as materials to stimulate bone-formation andbone-consolidation.

Chitosan is a kind of polysaccharide obtained by deacetylation ofchitin, an exoskeleton-structure material of sea Crustacea (Kind, G. M.,Bind, S. D., Staren, E. D., Templeton, A. J. and Economou, S. G., Curr.Surg., 47: 37, 1990; Hauschks, P. V., Bone, Vol. 1, 103, 1990, LondonCRC press; Cunningham, N. S., Paralkar, V. and Reddi, A. H., Proc. Nat.Acad. Sci., 89: 11740, 1982; Malette, W. G., Quigley, H. J. and Adickes,E. D., Nature and Technology, 435: 1986, New York Plenum Press).Muzarelli, etc have disclosed that chitosan introduced to bone-deficientregion stimulated normal bone-formation (Muzzarelli, R. A.,Mattioli-Belmonte, M., Tiets, C., Biagini, R., Feioli, G., Brunelli, M.A., Fini, M., Giardino, R., Ilari, P. and Biagini, G., Biomaterials, 15:1075, 1994), and Klokkevold, etc also have reported that chitosanstimulated differentiation of bony osteogenesis cells and inducedbone-formation itself (Klokkevold, P. R., Vandemark, L., Kenney, E. B.and Bernard, G. W., J. Periodontol., 67: 1170, 1996).

TGF-β is known to evoke proliferation and differentiation ofosteoblasts, especially β ig-h3 is believed to increase the productionof various bone intracellular proteins in vitro and decrease thecollagen degradation in osteoblasts (Sporn, M. B., Roberts A. B.,Springer-Verlag, New York: 3, 1990; Centrella, M., McCarthy, T. L. andCanalis, E., J. Bone Join. Surg., 73(Am): 1418, 1991; Mustoe, T. A.,Pierce, G. F., Thomason, A., Gramates, P., Sporn, M. B. and Deuel, T.F., Science, 237: 1333, 1987; Noda, M. and Camilliere, J. J.,Endoclinol., 124: 2991, 1989; Joyce, M. E., Jinguski, S., Roberts, A.B., Sporn, M. B. and Bolander, M. E., J. Bone Miner. Res., 4: 225, 1989;Hock, J. M., Canalis, E. and Centrella, M., Endoclinol., 126: 421, 1990;Beck, L. S., Ammann, A. J., Aufdemorte, T. B., DeGuzman, L., Xu, Y.,Lee, W. P., McFatridge, L. A. and Chen, T. L., J. Bone Miner. Res., 6:961, 1991). Among many growth factors, transforming growth factor β(TGF-β) is particularly important regulator for bone-regeneration anddevelopment. TGF-β 1 is a strong chemoattractant of osteoblast and leadsdivision of proto-cells of osteoblasts during the endochondrialossification process. As a cell-attaching protein whose expression isinduced by TGF-β, β ig-h3 has functions of attaching and spreading cellsby working with integrin (Jung-Eun Kim, Song-Ja Kim, Byung-Heon Lee,Rang-Woon Park, Ki-San Kim and In-San Kim, J. Biol. Chem., 275:30907-30915, 2000) as well as curing a cut. It is also known to play animportant role in the early phase of osteogenesis (Dieudonne, S. C.,Kerr, J. M., Xu, T., Sommer B., DeRubeis, A. R., Kuznetsov, S. A., Kim,I-S., Robey, P. G., and Young M. F., J. Cell. Biochem., 76: 231-243,1999).

Bone morphogenic protein (referred as “BMP” hereinafter) is abone-forming material and was first found by Urist. BMP has beenreported to stimulate pluripotential cells to be differentiated intochondrocytes and osteogenesis cells, and also play an important role inbone-regeneration (Urist, M. R., Science, 150: 893, 1965; Urist, M. R.and Strates, B. S., J. Dent. Res., 50: 1392, 1971; Wozney, J. M.,Butterworth Heinermann 1st Ed. London: 397-411, 1994; Wozney, J. M.,Mol. Reprod. Dev., 32; 160, 1992; Wozney, J. M., Rosen, V. and Celeste,A. J., Science, 242: 1528, 1988; Ono, I., Tatashita, T., Takita, H. andKuboki, Y., J. Craniofac. Surg., 7: 418, 1996). 13 human BMPs have beenconfirmed so far and especially human bone morphogenic protein-4 (BMP-4)has been reported to have excellent effect on bone-regeneration (Boyne,P. J., Bone, 19: 83s, 1996; Zegzula, H. D., Buck, D. C., Brekke, J.,Wozney, J. M. and Hollinger, J. O., J. Bone Join. Surg., 79: 1778, 1997;Sporn, M. B., Roberts A B (eds.), Springer-Verlag, New York: 3, 1990).

Tripolyphosphate is immediately hardened when water-soluble chitosan isadded thereto. Thus, it is required in this invention to injectchitosan, water soluble chitosan containing β ig-h3, a material tostimulate bone-formation and bone-consolidation, or bone morphogenicprotein simultaneously into the same spot using dual syringe shown inFIG. 2 in order to induce immediate solidation at the spot, resulting inpreventing injected materials from being transferred to other areas.

For the composition for stimulating bone-formation andbone-consolidation of the present invention prepared by adding β ig-h3or bone morphogenic protein as a material to stimulate bone-formationand bone-consolidation to the mixture of tripolyphosphate and watersoluble chitosan, tripolyphosphate and water soluble chitosan arepreferably mixed in the proportion of 20:80˜80:20 weight % and morepreferably 50:50 ratio. As a material to stimulate bone-formation andbone-consolidation, β ig-h3 is preferably added to the composition withthe amount of 100 μg/ml˜1 μg/ml and more preferably with 300 μg/ml˜600μg/ml. BMP is also preferably added to the composition with the amountof 50 ng/ml˜500 ng/ml and more preferably with 100 ng/ml˜300 ng/Ml.

The present invention also provides a use of the composition forstimulating bone-formation and bone-consolidation in the early phase.

In order to confirm whether the composition can be used for stimulatingbone-formation and bone-consolidation, the present inventors firstinvestigated the effect of the composition prepared by adding watersoluble chitosan, water soluble chitosan containing β ig-h3 or watersoluble chitosan containing BMP to tripolyphosphate on bone-formationand bone-consolidation as bone-extension operation was performed at thejaw of a dog.

As a result, in the bone sample of control group obtained 4 weeks and 7weeks after bone extension, extended area was proved to be solid but alittle flexible when being bended. As for BMP-4 group prepared by addingwater soluble chitosan containing BMP-4 to tripolyphosphate, β ig-h3group prepared by adding water soluble chitosan containing β ig-h3 totripolyphosphate and chitosan group prepared by adding just watersoluble chitosan to tripolyphosphate, extended bone samples taken 4weeks after bone extension were proved to be more solid than that ofcontrol group taken 7 weeks after bone extension. 7 weeks later, newbones were formed and the extended areas became very solid in thosegroups.

Radio-assay was performed for control group 4 weeks and 7 weeks afterbone extension. As a result, wide radiolucent zone was found betweenextended jaw-spicules and radiodense zone abutting on jaw-spicules washardly generated. The result obtained 4 weeks after bone extension wasnot very different from that obtained 7 weeks later in a control group.In the meantime, calcification was clearly observed at the extended areaof jaw-bone wherein bone growth materials were introduced in BMP-4group, β ig-h3 group and chitosan group. 4 weeks after introducing bonegrowth materials, it was confirmed by radio-assay that radiolucent zonebetween extende jaw-spicules was almost connected with radiodense zonegrowing from both sides of spicules. The thickness from top to bottom ofradiodense zone became more than two fold 7 weeks later, comparing tothat after 4 weeks. Especially, darker radiodense shadow and thickerradiodense zone were observed in the BMP-4 group, comparing to othergroups (see FIG. 4-FIG. 7).

Besides, every group had higher bone-mineral density than control group.Especially, BMP-4 group showed the highest bone-mineral density and βig-h3 group, chitosan group and control group followed in order.Bone-mineral density reflects radiodense level on extended area betweenjaw-spicules, meaning that the higher the density is the greater newbone-formation becomes (see FIG. 8).

Histological test was also performed. As a result, the wholebone-extended area was filled with fibrous tissue 4 weeks later incontrol group. Although new bone-formation close to jawbone section wasbegun by periosteum reaction, general bone-formation was not found (seeFIG. 9A). 7 weeks later, however, new bone and cartilage-formation weredetected near the edge of extended area and blood vessels and nervoustissues were also found in many places (see FIGS. 9B and 9C).

As for BMP-4 group, the proliferation of osteoblasts forming osteoid waspartly observed in center and near the edge of extended spicules, butmost parts of the extended area were filled with fibrous tissues 4 weekslater (see FIGS. 10A and 10C). 7 weeks later, irregular woven bonetrabeculae was partially calcified at extended area, newly formed bonearea wherein blood vessels in various sizes were spread and narrowfibrous interzone lying in the middle of the newly formed bone area wereobserved. The new bone formed throughout the whole extended area wassimilar to the normal cortical bone (see FIGS. 10B and 10D).

As for β ig-h3 group, osteoblasts forming osteoid were partly observedin the center of the extended area, which was confirmed by histologicaltest 4 weeks after bone-extension (see FIG. 11A). 7 weeks later, lots ofosteoblasts were partly forming new bone from the edge to the center ofextended area. The amount of newly formed bone was smaller than that ofBMP-4 group but fibrous interzone locating in the center of extendedarea was wider than that of BMP-4 group (see FIG. 11B and 11C).

As for chitosan group, histological test 4 weeks later confirmed thatmost extended area were filled with fibrous tissues (see FIG. 12A). 7weeks later, lots of osteoblasts along with new bone were observed overthe edge of extended area and new bones were confirmed to be formedpartly form the edge to the center of extended area (see FIGS. 12B and12C). The amount of newly formed bone in extended area was smaller thanthat of β ig-h3 group or BMP-4 group, but fibrous interzone was widerthan that of β ig-h3 group.

Resultingly, the composition of the present invention which was preparedby adding water soluble chitosan, water soluble chitosan containing βig-h3, and water soluble chitosan containing BMP-4 to tripolyphosphatecan be effectively used for stimulating bone-formation andbone-consolidation by stimulating bone-formation and bone-consolidationin the early phase and by shortening bone-consolidation period.

EXAMPLES

Practical and presently preferred embodiments of the present inventionare illustrative as shown in the following Examples.

However, it will be appreciated that those skilled in the art, onconsideration of this disclosure, may make modifications andimprovements within the spirit and scope of the present invention.

Example 1 Preparation of Composition

The present inventors have prepared a composition for stimulatingbone-formation and bone-consolidation by adding a material to stimulatebone-formation and bone-consolidation to tripolyphosphate. Particularly,the present inventors prepared a composition by adding 0.5 ml of 5%chitosan to 0.5 ml of 5% tripolyphosphate and named it “chitosan group”.

Example 2 Preparation of Composition

The present inventors prepared a composition for stimulatingbone-formation and bone-consolidation by adding 0.5 Ml of water solublechitosan containing β ig-h3 at the concentration of 450 μg/ml to 0.5 mlof 5% tripolyphosphate with the same method as the above Example 1 andnamed it “β ig-h3 group”.

Example 3 Preparation of Composition

The present inventors prepared a composition for stimulatingbone-formation and bone-consolidation by adding 0.5 ml of water solublechitosan containing BMP-4 at the concentration of 200 ng/ml to 0.5 ml of5% tripolyphosphate with the same method as the above Example 1 andnamed it “BMP-4 group”.

Experimental Example 1 Investigating the Effect of the Compositions ofthe Present Invention on Bone-Formation and Bone-Consolidation

In order to confirm whether the compositions for stimulatingbone-formation and bone-consolidation prepared through Example 1-Example3 of the present invention can stimulate new bone-formation andbone-consolidation in the early phase, the present inventors firstperformed bone extension operation at jawbones of dogs, followed byinjecting the compositions for stimulating bone-formation andbone-consolidation thereto, and then observed the changes occurring.

Particularly, used 16 5-8 month old dogs for experiments and groupedthem by 4 for control group, chitosan group, β ig-h3 group and BMP-4group.

Kept the breath of dogs through tubes inserted in organs after generalanesthesia and shaved the operating part, followed by sterilization andapplication. Incised skin 3-4 cm along the lower end of jawbone, liftedmasseter muscle and exposed the side part of jawbone. Then, performedvertical osteotomy at the trunk of jawbone using an electric saw andcompletely cut jawbone. Fixed each fixing pin of external fixator onspicule 1 cm away from the cutting area right and left. While fixing thepins on the jawbone spicule with drill, kept washing with salinesolution not to burn the fixing sites. Inserted the pins just as deep asit barely passed through jawbone and then fixed them tightly. Afterfixing two pins all, set them on bone extending apparatus (MolinaDistractors, Wells Johnson Company) (FIG. 1).

Sutured the incised area with 5-0 vicryl and 5-0 nylon stitching fiberlayer upon layer and recovered the dogs from anesthesia. Administeredpenicillin (100,000μ/kg) by intramuscular injection every 12 hours for 7days after operation and oral-administered anodyne every 4-6 hours torelieve pain. Fed the dogs with soft diet until the second day afteroperation and provided regular diet from the third day. From the fifthday after operation, started bone-extension 2 mm per day for 5 days (upto 10 mm total).

On the day when bone extension was finished, injected 5% water solublechitosan, 5% water soluble chitosan containing BMP-4(200 ng/ml) (R&DSystem Inc.) and 5% water soluble chitosan containing β ig-h3 (450μg/ml) with the same amount as 0.5 ml of 5% tripolyphosphate(TPP)simultaneously to the same extended spot using a dual syringe (FIG. 2)for each group to induce immediate hardening by mixing those materials,resulting in the prevention of transferring those injected materials toother areas for strong new bone formation. On the contrary, injectedonly 1 ml of 5% tripolyphosphate to a control group.

Upon completing the injection of compositions for stimulatingbone-formation and bone-consolidation of the present invention to thedogs, kept bone-extending apparatus on them for 7 weeks forbone-consolidation and bone-regeneration. Sacrificed every 2 dogs fromeach group by injecting overdose of pentobarbital (40-50 mg/kg) 4 weeksafter bone extension, so did for the rest 8 dogs 7 weeks after boneextension (FIG. 3).

<1-1> Observation with the Naked Eye

The changes after the above experiments were observed with the nakedeye. Every dogs were well recovered from anesthesia and operation,bone-extending apparatus was kept stable and no signs of infectionaround the apparatus were shown. Bone samples taken 4 weeks and 7 weeksafter bone extension confirmed that the extended bone area was hardenedbut a little flexible when being bended in a control group. Meanwhile, 4week old extended bone areas of BMP-4 group, β ig-h3 group and chitosangroup were harder than 7 week old extended bone area of a control groupand all the groups except the control group showed very strong and solidnew bone formation 7 weeks later.

<1-2> Radio-Assay

Examined each animal group went through bone extension with radio-assayevery week and observed bone-formation and bone-consolidation throughthe radiographs taken after 4 weeks and 7 weeks respectively.

As a result, wide radiolucent zone between extended jawbone spicules wasseen in a control group both 4 weeks and 7 weeks after bone extensionwas completed while radiodense zone close to both sides of jawbonespicules was hardly seen 4 weeks later having no difference in resultobtained 7 weeks after bone extension. In the meantime, calcificationwas undergoing with the lapse of time in extended area of jawbone inevery BMP-4 group, β ig-h3 group and chitosan group whereto appropriatematerial for bone growth was injected. As for those groups, radiolucentzone in between extended spicules was almost connected with radiodensezone growing from each side of spicules, which was confirmed byradiographs taken on the 4^(th) week. On the 7^(th) week, the thicknessof radiodense zone became two-fold comparing with that of the 4^(th)week. Especially, the density of radiodense shadow in BMP-4 group wasthicker and darker comparing to other groups.

<1-3> Measurement of Bone Mineral Density

Based on radiographs (FIG. 4B, 5B, 6B and 7B) taken on the 7^(th) weekin the above Example <1-2>, the present inventors measured bone mineraldensity with computer program by taking advantage of the fact thatradiodense zone becomes brighter on radiograph as bone-formationprogresses.

As a result, bone mineral density in those groups was higher than thatin control group on the 4^(th) week. Especially, BMP-4 group showed thehighest density and β ig-h3 group, chitosan group and control groupfollowed in order (FIG. 8A). BMP-4 group still showed remarkably highdensity comparing to other groups on the 7^(th) week and β ig-h3 group,chitosan group and control group also followed in order (FIG. 8B). Bonemineral density reflects the brightness on a radiograph, in other words,the degree of radiodense in extended area between jawbone spicules.Thus, the higher the density, the greater the amount of bone-formationis. Resultingly, it was confirmed that the groups whereto compositionsfor stimulating bone-formation and bone-consolidation were added showedfaster and greater bone-formation than a control group.

<1-4> Histological Test

Bone samples were taken from extended jawbone including normal osseoustissues therearound with an electric saw. The obtained spicules werefixed in 10% neutral formalin for 1 week, and then decalcified in 10%nitric acid and 10% sodium citrate for 2 days. 4-6 μm of specimens wereprepared by dehydration and paraffin-fixation following generaltechniques. The specimens were stained with hematoxylin-eosin to observehistological changes with an optical microscope.

As a result, it was observed in the control group that the wholeextended bone area was filled with fibrous tissues and general newbone-formation was not detected even though new bone-formation byperiosteum reaction had just begun along the section of jawbone on the4^(th) week after bone extension (FIG. 9A). New bone and cartilage wereformed over the edge of extended area and blood vessels and nervoustissues were also observed in many places (FIGS. 9B and 9C).

As for BMP-4 group, although the proliferation of osteoblasts formingosteoid was partly observed in the center and through the edge ofextended spicules, most parts of the extended area were filled withfibrous tissues on the 4^(th) week (FIGS. 10A and 10C). On the 7^(th)week, widely formed new bone area having irregular woven bone trabeculaeresulted from partial calcification, various sized blood vessels andnarrow fibrous interzone lying up and down in the center of new bonearea were observed. The new bone formed throughout the whole extendedarea was almost similar to the normal cortical bone (FIG. 10B and 10D).

As for β ig-h3 group, it was observed on the 4^(th) week thatosteoblasts forming osteoid were partly proliferated in the center ofthe extended area (FIG. 11A). On the 7^(th) week, many activeosteoblasts were partly forming new bone from the edge through thecenter of the extended area, but the volume of new bone was smaller thanthat of BMP-4 group. The fibrous interzone lying up and down in thecenter of the extended area was, though, wider than that of BMP-4 group(FIG. 11B and 11C).

As for chitosan group, most parts of the extended area were filled withfibrous tissues on the 4^(th) week (FIG. 12A). On the 7^(th) week, lotsof osteoblasts along with new bone were observed over the edge of theextended area and also new bone was partly formed from the edge throughthe center of the extended area. The volume of newly formed bone wassmaller than those of β ig-h3 group and BMP-4 group but the fibrousinterzone was wider than that of β ig-h3 group (FIGS. 12B and 12C)

Resultingly, as for BMP-4 group, β ig-h3 group and chitosan grouptreated with the composition for stimulating bone-formation andbone-consolidation of the present invention, new bone was partly formedin extended area on the 4^(th) week after bone extension and theformation went further on the 7^(th) week. BMP-4 group had the biggestvolume of newly formed bone and β ig-h3 group, chitosan group followedin order. Fibrous interzone was found in the center of the extended areain every group on the 7^(th) week. The fibrous interzone of BMP-4 groupwas the narrowest, β ig-h3 group showed the second narrowest fibrousinterzone and chitosan group was the last. In the meantime, the fibrousinterzone took most parts of the extended area in the control group.Observing fibrous interzone still on the 7^(th) week suggests thatnew-bone formation is still undergoing.

INDUSTRIAL APPLICABILITY

As described hereinbefore, the composition of the present inventionprepared by adding a material for stimulating bone-formation andbone-consolidation to the mixture of tripolyphosphate and water-solublechitosan can be effectively used for stimulating bone-formation andbone-consolidation. Precisely, the composition induces newbone-formation, provides a normal bone-structure, prevents the growth ofunnecessary connecting tissues and is suitable enough for human tosupplement bone-loss during the recovery process as well as induces thegrowth of blood vessels and bony osteogenesis cells in the early stage.

1. A composition containing tripolyphophate and water-soluble chitosanfor stimulating bone-formation and bone-consolidation.
 2. Thecomposition for stimulating bone-formation and bone-consolidation as setforth in claim 1, wherein the composition could additively contain amaterial for stimulating bone-formation and bone-consolidation.
 3. Thecomposition for stimulating bone-formation and bone-consolidation as setforth in claim 2, wherein the material for stimulating bone-formationand bone-consolidation is selected from a group consisting of β ig-h3,bone morphogenic protein, TGF-β, FGF, IGF-1 and PDGF.
 4. The compositionfor stimulating bone-formation and bone-consolidation as set forth inclaim 1, wherein the ratio of tripolyphosphate to water-soluble chitosanis 20:80˜80:20 weight %.
 5. The composition for stimulatingbone-formation and bone-consolidation as set forth in claim 4, whereinthe ratio of tripolyphosphate to water-soluble chitosan is 50:50 weight%.
 6. The composition for stimulating bone-formation andbone-consolidation as set forth in claim 3, wherein the β ig-h3 is addedat the concentration of 100 μg/ml˜1 μg/ml.
 7. The composition forstimulating bone-formation and bone-consolidation as set forth in claim3, wherein the bone morphogenic protein is added at the concentration of50 ng/ml˜500 ng/ml.
 8. The composition for stimulating bone-formationand bone-consolidation as set forth in claim 6, wherein the β ig-h3 isadded at the concentration of 300 μg/ml˜600 μg/ml.
 9. The compositionfor stimulating bone-formation and bone-consolidation as set forth inclaim 7, wherein the bone morphogenic protein is added at theconcentration of 100 ng/ml˜300 ng/ml.
 10. The composition forstimulating bone-formation and bone-consolidation as set forth in claim4, wherein the bone morphogenic protein is BMP-4.